Regulation of Eukaryotic and Prokaryotic Protein Kinases

Protein kinases transfer the gamma-phosphate of ATP to the hydroxyl groups of specific serine, threonine (serine/threonine kinases) or tyrosine (tyrosine kinases) residues in substrate proteins. This covalent modification of the Ser/Thr/Tyr–OH moiety is a fundamental mechanism of cellular regulation and intracellular signal transduction in eukaryotes and has a significant role in almost every aspect of cell growth, differentiation, maturation, motility and regulated cell death in eukaryotes. While crystallographic analyses have provided a weatlth of information about these key signalling molecules, the remaining key questions seem best answered by solution NMR spectroscopy. Solution NMR analyses of eukaryotic protein kinases are hindered both by the limited ability to obtain sufficient amounts of properly folded, soluble protein labeled with NMR-active isotopes and by the limitations of conventional NMR techniques due to the large size and limited ranges of concentrations and solution conditions available. Using a combination of molecular biology, biochemistry and novel NMR methodology we are studying the structure, dynamics and interactions of representative protein kinase subfamilies.
Tyrosine phosphorylation, long recognized as a central signaling mechanism in higher eukaryotes, is beginning to emerge as an important mechanism in prokaryotic signal transduction and has been shown to play a central role in bacterial pathogenesis. Towards our long term goal of assessing the functional implications of the transfer of just 4 atoms comprising the γ-phosphate group of ATP to specific tyrosine residues on key protein targets across the three domains of life, we have recently started work on a unique bacterial tyrosine kinase Wzc that has been shown to be cruicial in the synthesis and export of polysaccharides that are involved in biofilm or capsule formation.
Collaborators: Kevin Dalby (University of Texas, Austin), Christophe Grangeasse (IBCP, CNRS, Lyon, FRANCE)

Many viruses contain RNA genomes and utilize a multi-protein polymerase complex (PX) for RNA replication and transcription, processes that are central to viral infectivity, survival and propagation. In bacteriophages of the family cystoviridae (cystoviruses) that contain a double-stranded RNA (dsRNA) genome, the PX is a four-protein complex that includes an RNA-directed RNA polymerase (P2) that replicates and transcribes viral RNA (vRNA) in concert with proteins P1, P4 and P7. A complete understanding of the functional interactions of the PX proteins in spatial and temporal terms at atomic or near-atomic resolution is important not only in the context of cystoviruses in particular but for dsRNA viruses in general. This includes the more complex reoviruses that are far less amenable to in vitro and in vivo manipulations using molecular virology tools. We are attempting to answer these questions using a combination of several biophysical techniques including solution NMR and X-Ray crystallography.
Collaborator: Craig Cameron (Penn State University)

It is now accepted that in addition to static structures, the elucidation of dynamics is essential in order to understand fully the functional regulation of proteins. Solution-state nuclear magnetic resonance (NMR) reports structural as well as dynamic information over wide range of timescales and therefore has the potential to provide a clear description of proteins both in space and time. Our research involves the development of novel NMR methods to probe the dynamics nuclei of both the protein backbone and sidechains on mutiple timescales.
Collaborator: Fabien Ferrage (ENS, CNRS, Paris, FRANCE)

Nick Recognition and Repair by DNA Ligases

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3’-OH/5’-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Using a minimal eukaryotic DNA ligase (Paramecium bursaria Chlorella Virus ligase - ChVLig) as a model, we are investigating the process of nick recognition and repair by these key enzymes.
Collaborator: Stewart Shuman (Sloan-Kettering Institute)